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Who’s afraid of the big bad plague? Bet you the owners of the bones above were. This Halloween, we’re highlighting the work of researchers who tested (extremely carefully) for the presence of the pathogen Yersinia (Y.) pestis (a.k.a., the “Bubonic Plague”) in human skeletal remains from three sites in Germany and Switzerland. Thought to be victims of the Black Death, these individuals died alongside an estimated 75-200 million other Europeans affected by this outbreak of the plague, which diminished Europe’s population during the 14th century by one third.

The specialized protocol the researchers used in this study was carefully created to be in line with modern plague diagnostic procedures and to address the unique challenge of working with ancient DNA (aDNA), which varies in quality from sample to sample and is easily contaminated with modern DNA. Contamination is common and can be hard to identify, often resulting from poor handling practices at excavation or during preparatory procedures. The researchers did their best to avoid the possibility by thoroughly cleaning the surfaces of the bones and teeth that samples were drawn from, and using multiple controls to highlight any points of contamination from the laboratory that occurred during the experiments. At a time when the results from testing of aDNA samples can be highly contested, a validated DNA replication process was used to ensure authenticity of the tests and to prevent misinterpretation of the results by the scientific community.

Bones and teeth from 29 individuals, ranging from 300-600 years old, were collected from sites in Manching-Pichl and Brandenburg in Germany and Basel, Switzerland and housed at the State Collection for Anthropology and Palaeoanatomy in Munich. Selected samples were then moved to newly constructed labs at the ArcheoBio Center of the Ludwig Maximilian University Munich for preparation and aDNA extraction. Researchers followed a strict protocol to prevent any sample contamination at this stage. The new facility contains three air-locked and pressurized rooms, each meant to provide a contamination-free workplace for processing aDNA samples for replication. Before admittance to the three-room complex, staff were required to shower, wash their hair, and enter a gowning room to replace their freshly laundered clothes with two pairs of gloves, a hairnet, hooded overalls, and a screened facemask. A second gowning room required the addition of another set of hooded overalls. Scientists then moved through the rooms sequentially, preparing the aDNA samples and negative controls (meant to test for contamination in the replication process) for analysis.

Once preparatory procedures were complete, sealed tubes containing the negative controls and aDNA were transferred to the Bundeswehr Institute of Microbiology for the addition of positive controls (tubes containing DNA from Y. pestis), DNA replication and analysis. Of the 29 samples tested, seven contained fragments of a Y. pestis gene after an initial round of replication, and four additional samples tested positive for Y. pestis after further rounds of testing.

Although the skeletons above are not, Y. pestis is still alive and well in parts of the world. Now called the “Modern Plague” to differentiate it from previous plagues caused by the same pathogen, such as Justinian’s plague and the Black Death, the disease affects 1,000-3,000 people per year. Modern treatments have thankfully limited the number of deaths that result from these cases, leaving us less likely to end up in the ground after getting sick, like these poor individuals. Nevertheless, research on the presence of the pathogen in ancient samples remains crucial for our continued understanding of how this disease affected our population in the past.

So, in case you need a scary costume idea for tonight’s festivities, why not draw some inspiration from our friends above? A skeletal Black Death victim and a masked, double-overalled plague researcher sound like great costume ideas to us.